Proteomic analysis of Bacillus anthracis Sterne vegetative cells

Biochimica et Biophysica Acta 1748 (2005) 191 – 200 http://www.elsevier.com/locate/bba

Proteomic analysis of Bacillus anthracis Sterne vegetative cells Anthony W. Francisa,1,2, Christy E. Ruggierob,1, Andrew T. Koppischa, Jingao Donga, Jian Songa, Thomas Brettina, Srinivas Iyera,T a

Bioscience Division, Los Alamos National Laboratory, P.O. 1663, Los Alamos, NM 87545, United States Chemistry Division, Los Alamos National Laboratory, P.O. 1663, Los Alamos, NM 87545, United States

b

Received 3 November 2004; received in revised form 3 January 2005; accepted 13 January 2005

Abstract Mass spectrometry and proteomics have found increasing use as tools for the rapid detection of pathogenic bacteria, even when they are in a mixture of non-pathogenic relatives. The success of this technique is greatly augmented by the availability of publicly accessible proteomic databases for specific pathogenic bacteria. To aid proteomic detection analyses for the causative agent of anthrax, we have constructed a comprehensive proteomic catalogue of vegetative Bacillus anthracis Sterne cells using liquid chromatography tandem-mass spectrometry. Proteins were separated by molecular weight or isoelectric point prior to tryptic digestion. Alternatively, the whole protein extract was digested and tryptic peptides were separated by cation exchange chromatography prior to Reverse Phase-LC-MS/MS. The use of three complementary, pre-analytical separation techniques resulted in the identification of 1048 unique proteins, including 694 cytosolic, 153 membrane (including 27 cell wall), and 30 secreted proteins, accounting for 19% of the total predicted proteome. Each identified protein was functionally categorized using the gene attribute database from TIGR CMR. These results provide a large proteomic catalogue of vegetative B. anthracis cells and, coupled with the recent proteomic catalogue of B. anthracis spore proteins, form a thorough summary of proteins expressed in the active and dormant stages of this organism. D 2005 Elsevier B.V. All rights reserved. Keywords: Bacillus anthracis Sterne; Proteomics; Mass spectrometry

1. Introduction Bacillus anthracis is the causative agent of the disease anthrax [1–3], and is currently the subject of a great deal of research both into strategies for combating infection, as well as its detection in the environment. World events have illustrated that the potential release of anthrax as a terrorist weapon is a serious threat. Many avenues for the detection of B. anthracis are under current investigation, however, all of them are confounded by the observation that pathogenic B. anthracis is nearly genetically identical to a number of other widespread, non-pathogenic bacilli [4]. Thus, detecting B. T Corresponding author. MS M888, P.O. 1663, Los Alamos, NM 87545, United States. Tel.: +1 505 665 5122; fax: +1 505 665 3024. E-mail address: [email protected] (S. Iyer). 1 AWF and CER contributed equally to this paper. 2 Current address. Silver State Analytical Laboratories, Las Vegas, Nevada, 89118, United States. 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.01.007

anthracis in the environment, distinguishing it from its nonpathogenic relatives, and doing so in a rapid manner to provide adequate time to facilitate containment in the event of a bioterror release remain a daunting task. Mass spectrometric techniques have shown great promise in the ability to accurately identify vegetative B. anthracis from within a bacterial cell mixture rapidly [5,6] as well as the ability to characterize spore species [7]. The availability of a proteome database is critical for bacterial identification [8] using these techniques and the recent proteomic catalogue of the B. anthracis var Sterne spore [9,10] has greatly augmented these efforts. However, at this time, no proteomic profile for this organism exists for its vegetative state. Although spores are the infective form of the organism [3], its vegetative form exists in the environment and has also been observed to be present in food sources contaminated with B. anthracis [11,12]. Thus a proteomic profile of the vegetative form is also desirable to augment existing detection techniques.

192

A.W. Francis et al. / Biochimica et Biophysica Acta 1748 (2005) 191–200

The sequencing and annotation of the B. anthracis Ames genome revealed the presence of 5838 genes distributed over the chromosome (5508 genes) and two virulencerelated plasmids, pXO1 (204 genes) and pXO2 (104 genes) [13]. More recently, the Sterne genome (5491 genes) has been sequenced by the Joint Genome Institute (GenBank Accession # AE017225) and the accessibility of this genomic data has allowed us to conduct a comprehensive proteomic study. In this study, we have focused on cataloging the B. anthracis Sterne proteome in the vegetative state, which may provide additional insight into targets for vaccine development. Combinations of separation methods and mass spectrometry-based proteomics have increasingly become the preferred method for analyzing large, complex protein samples. Here, we used three discrete pre-analytical separations followed by peptide identification by LC-MS/ MS and database searching.

with phosphate-buffered saline, yielding 8 g of wet cells. The cell pellet was resuspended and washed in 50 mM Tris buffer, pH 7.2, and collected by centrifugation at 4000g for 10 min at 4 8C. Protein extraction was performed using the PROT-MEM kit (Sigma) and concentration was determined using the BCA assay (Pierce).

2. Materials and methods

2.3. Isoelectric focusing

2.1. Bacterial cultures

IEF separations were performed as described previously [14]. Briefly, 50 Ag of each protein fraction was added to the rehydration buffer (8 M urea, 2% CHAPS, 10 mM DTT, 2% carrier ampholytes, 0.01% bromophenol blue), and 17 cm, pH 3–10, Immobilized pH Gradient (IPG, Bio-Rad, Richmond, CA) strips were passively rehydrated overnight at room temperature. The next day, focusing was performed at 20 8C on a Protean IEF Cell (Bio-Rad) using a rapid voltage slope and a 500 V hold step at the end of the run (60,000 V hours total). Each IPG strip was cut into 24 equally sized segments and subjected to in-gel tryptic digestion (described later).

B. anthracis Sterne strain 7702 (pXO2 ) was originally obtained from Paul Jackson at Los Alamos National Laboratory. Cultures were grown overnight at 30 8C in 5 ml of brain–heart infusion medium (BHI, Difco Laboratories) with shaking. In order to minimize the carryover of the BHI media, 0.5 mL of this culture was used to inoculate 25 mL Fe-free CA media {Per L: 3.6 g Cas amino acids, 100 mM pH 8 HEPES, 5 mM KH2PO4, 5 mM K2HPO4, 200 mM Thiamine HCl, 200 mM glycine, 70 mM l-cysteine, 400 mM l-tryptophan, 15.5 mM adenine, 12.5 mM uracil (all chelexed), 100 mM CaCl2, 40 mM MgSO4, 5 mM MnSO4 (adjust pH to 7.8 and autoclave entire solution), then added 10 mL of 20% glycerol (chelexed and sterile filtered) and 125 mL trace metals (sterile filtered). Trace metals per 100 mL: 7 mg CuSO4, 3.5 mg MnSO4d H2O, 2.4 mg ZnCl3, 100 mg CaCl 2 , 1.8 mg CoCl 2 , 0.70 mg H 3 BO 4 , 6 mg (NH4)6Mo7O24d 4H2O}, which, after growth to O.D. N1, was then passed again through Fe-free CA media (1.5 mL into 75 mL). It is standard procedure in our laboratory to grow all pass-through cultures in a Fe-free CA media, as some of our applications for these cultures are iron-sensitive. Nevertheless, we do not expect this to influence our results, as the cells harvested for this analysis were ultimately cultured in the presence of iron. This culture was grown to an O.D. of 0.7, and used to inoculate CA media containing 30 AM Fe (as Fe-tiron 1:3 chelate, 12 mL inoculum into 2.5 L). This culture was grown with vigorous shaking (27 8C, 100 rpm) under ambient atmosphere until mid to late log phase (12–16 h). Ambient temperature and CA media (minimal nutrients) were selected to best represent the conditions in which vegetative cells would be found in the environment. Cells were harvested by centrifugation and washed twice

2.2. SDS-PAGE 100 Ag of extracted protein was mixed with of SDS loading buffer (1:2 sample to loading buffer volumes). The sample was heated to 95 8C for 15 min and loaded into 1 mm thick, 10 cm long, 12% SDS-polyacrylamide gel. Electrophoresis was performed for 1.5 h at 200 V or until the dye front migrated to the bottom of the gel. Post-separation, the gel was stained using Sypro Ruby Protein Stain (BioRad) and each lane was cut into 12 equally spaced slices. Each slice was subsequently minced into 1 mm3 cubes for tryptic digestion (described later).

2.4. Off-line two-dimensional peptide chromatography 300 Ag of protein from total lysate was digested with trypsin (in solution, described later) and peptides were loaded onto a cation exchange column cartridge (ABI) using a 3 ml syringe. Fractions (500 AL) were collected by eluting with successive salt concentrations of 50, 100, 250, 500, and 1000 mM NaCl. Due to the high salt content, these fractions were first desalted with C18 cartridges (Millipore) prior to reverse phase LCMS/MS analysis. In parallel, total digests were also analyzed using RPLC-MS/MS. 2.5. Trypsin digestion Modified porcine trypsin (Promega) was used throughout. In-gel digestion was performed essentially as described by Shevchenko et al. [15]. Briefly, minced gel pieces (or IPG strip sections) were washed with 100 mM ammonium bicarbonate (AMBIC), dehydrated with acetonitrile, dried in a Speedvac (Savant) and then subjected to a reduction and alkylation treatment with 10 mM DTT (56 8C for 30 min)

A.W. Francis et al. / Biochimica et Biophysica Acta 1748 (2005) 191–200

and 55 mM Iodoacetamide (RT in dark for 30 min) respectively. Following two more washes with AMBIC and dehydration with acetonitrile, the gel pieces were incubated in trypsin digestion solution containing 12.5 ng/ AL trypsin in 50 mM AMBIC containing 5 mM CaCl2 on ice for 1 h. At the end of the hour, unabsorbed solution was removed and the same solution (without trypsin) was added to just cover the pieces and left overnight (16 h) at 37 8C. Resultant peptides were extracted by sonication in 50% acetonitrile/0.1% TFA. The peptides were dried down, resuspended in 0.1% TFA and desalted using C18 Ziptips (Millipore). For the total lysate digestion, pH was adjusted to 8.5 using AMBIC and concentration was estimated using the BCA kit (Pierce). Following reduction with DTT and alkylation with iodoacetamide, trypsin (final concentration of 10 ng/AL in 25 mM AMBIC) was added and digestion was carried out at 37 8C overnight. 2.6. High performance liquid chromatography A PepMap C18 column (3 Am, 100 2, 75-Am i.d.15 cm) (Dionex) was used within a LC Packings UltiMate microcapillary LC system. The mobile phases consisted of (A) 0.1% formic acid and 5% acetonitrile and (B) 0.1% formic acid and 95% acetonitrile. A 5 AL injection of each peptide sample was loaded onto the HPLC system for each analysis. After an initial 5 min period at 5% B, a linear gradient proceeds up to 50% B over 50 min ending with a step up to 75% B for 10 min. The microcapillary-LC system was run at 0.2 Al/min. 2.7. Tandem mass spectrometry The microcapillary LC system was coupled to a QSTAR XL nanospray-MS/MS (Applied Biosystems) through a 10 Am i.d. PicoTip nanospray emitter (New Objective, Woburn, MA) mounted on the nanospray source (Proxeon, Denmark). The electrospray voltage was set at 1.8 kV. Samples were analyzed using a data-dependent MS/MS mode and rolling collision energy was used to fragment the peptide ions based on the m/z value and charge state.

193

sequence verification. The functional classification of B. anthracis Sterne proteins was performed according to the TIGR CMR gene attributes for B. anthracis Ames (http:// www.tigr.org/tigr-scripts/CMR2/gene_table.spl?db=gba). The functional classifications of the proteins in Ames strain were applied to the proteins in Sterne when significant sequence similarity was found. 2.9. Protein location The cellular locations of B. anthracis Sterne proteins were predicted using Psort-B [16] in combination with additional sequence analyses using Prosite (http://us.expasy. org/prosite/) and Pfam (http://www.sanger.ac.uk/Software/ Pfam/). The presence of N-terminal signal sequences (both cleavable and uncleavable), lipoprotein signal, transmembrane domains, etc., are predicted by Psort and the presence of specific sequence features such as cell wall anchoring LPXTG motif and S-layer homology (SLH) domain are predicted by Prosite and Pfam. Proteins without such sequence features were categorized as cytosolic.

3. Results 3.1. Pre-analytical separations The total cellular proteins extracted from B. anthracis Sterne strain vegetative cells were identified using a combination of three distinct pre-mass spectrometric methods: (1) protein separation by molecular weight using SDSPAGE, (2) protein separation by pI using IEF on broad range IPG strips and (3) total tryptic peptide separation by ionic strength using strong cation exchange (SCX) chromatography. In-gel trypsin digests were performed following SDS-PAGE and IEF while a solution trypsin digest was performed prior to the cation exchange method. Although SDS-PAGE + RP-LCMS/MS

478

IPG + RP-LCMS/MS

93 127

2.8. Data processing and analysis 175

The LC-MS/MS data were analyzed against the B. anthracis Sterne database. Search parameters included 1 missed trypsin cleavage and selected possible post-translational modifications (carboxymethylation of cysteine, oxidation of methionine, and phosphorylation of serines, threonines, and tyrosines). Protein identifications were accepted based on ProID confidence scores (which is based on number of peptides, peptide scores and distance from nearest neighbor) of 90% or higher in accordance with software instructions from ABI and the list of proteins identified were classified based on confidence levels. Randomly selected sets were manually confirmed for peptide

10

57

75

Off-line 2D LCMS/MS Fig. 1. Venn diagram of the distribution of proteins identified by the three distinct pre-analytical separation methods. As shown, a total of 681 proteins (478, SDS-PAGE; 75, SCX; and 127, IEF) was only found in one of the three methods used. In addition 33 unique hits were obtained when a total digest was directly applied to RPLC-MS/MS and is not depicted in this Venn diagram.

194

A.W. Francis et al. / Biochimica et Biophysica Acta 1748 (2005) 191–200

Fig. 2. Comparison of hypothetical 2D gels of the proteins identified in this study (A, top panel) with the predicted B. anthracis proteome (B, bottom panel).

cutting gel fragments from IPG strips for use in in-gel trypsin digests is a relatively uncommon approach, we found the resulting peptide fractions to be of excellent quantity and quality. All fractions were analyzed by reversed phase microcapillary liquid chromatography (RPLC) coupled with nano-electrospray ionization tandem mass spectrometry (LC-MS/MS). 3.2. Mass spectrometric identification ProID software using the Interrogatork algorithm was used to search each MS/MS spectra against the newly created B. anthracis Sterne predicted proteome. The separation methods resulted in the identification of 1048 unique proteins (greater than N90% confidence) with an average confidence score of 97% and 2.9 peptides per

protein. The entire list of proteins identified in this study is included in the supplemental information. Many MS/MS spectra obtained did not result in the identification of B. anthracis proteins due to database search results that were below the selected threshold confidence level (90%) for a given hit. The SDS-PAGE method resulted in a total of 803 protein identifications, IEF produced 405, and SCX-RP method produced 317 (Fig. 1). An additional 249 proteins were identified when tryptic peptides of the total lysate were directly processed by RPLC-MS/MS, of which 33 were unique to this approach, however it is evident that performing SCX chromatography dramatically improves the subsequent analysis. The overlap between the methods reduced the final count to 1048 unique proteins, representing 19.1% of the predicted B. anthracis Sterne proteome. Each method produced a subset of unique proteins that were not found in the other two, demonstrating the effectiveness of using a combination of methodologies to increase the proteomic coverage of a given organism (Fig. 1). Although the Sterne genome is often assumed to be identical to the Ames genome, except for the deletion of the pXO2 plasmid, there are several point mutations that are present in the Sterne genome that can result in missed identifications during database searching. In fact, 8 proteins were identified using the Sterne database, which were not identified at N66% confidence when searched against the Ames database. Of these proteins, five (BAS0142, BAS0401, BAS1193, BAS2221, and BAS3743) were predicted in Sterne but not in Ames; two (BAS1025/BA1098 and BAS2317/BA2494) were predicted to have different translational start sites between Ames and Sterne and one (BAS1622/BA1748) is different as a result of copy number change in a DNA repeat (VNTR) within the coding region. In addition, 9 proteins were identified at N90% confidence using the Ames database which were not found at N66% confidence when searched against the Sterne database and are not included in the final reported results. Six of these (BA4834, BA4392, BA2595, BA2471, BA2023, and BA1547) are very short ORFs and were not predicted in Sterne although the nucleotide sequences are identical between the two strains and two (BA0816/BAS0778 and BA3789/BAS3512) were

Hypothetical proteins Energy metabolism Unknown function Protein synthesis Transport and binding proteins Cellular processes Cell envelope Regulatory functions Protein fate DNA metabolism Purines, pyrimidines, nucleosides, and nucleotides Biosynthesis of cofactors, prosthetic groups, and carriers Fatty acid and phospholipid metabolism Amino acid biosynthesis Central intermediary metabolism Transcription Other categories Fig. 3. Distribution of identified Sterne proteins as per functional categories.

A.W. Francis et al. / Biochimica et Biophysica Acta 1748 (2005) 191–200

predicted to have a different translational start site. One (BA2452) should probably have been found using the Sterne database (as BAS2283) since the predicted proteins are nearly identical. There was also one difference in gene prediction between the two strains; two ORFs (BA3628 and BAS3629) were predicted in Sterne while a single ORF (BA3915) was predicted in Ames although the nucleotide sequences are identical. These differences in gene prediction between Sterne and Ames caused discrepancies in the overall proteins assigned by the ProID algorithm for the two strains. In addition to the 5491 predicted ORFs in B. anthracis Sterne, we also looked at the potential ORFs that may have been missed in the intergenic regions. ORFs that are longer than 30 amino acids from the six frame translation of all of

195

the intergenic regions were used for the search. Two ORFs that were hit with high confidence (98%) were found. One (Genome coordinates 538255–537923 in Sterne and 538212–537881 in Ames) of the ORFs has no significant Blast hits to any known proteins in the GenBank and the other (747838–747527 in Sterne and 747942–747631 in Ames) has significant similarity to a hypothetical protein (gi47092471) in Listeria monocytogenes. 3.3. Proteomic analysis Contrary to our initial expectations, we did not notice a significant bias for any technique towards a particular class of proteins (data not shown), especially based on their

Table 1 Subset of identified proteins that may be related to virulence/pathogenesis Gene ID

gi #

Description

Loca

BAS0147 BAS0155 BAS0270 BAS0336 BAS0505 BAS0581 BAS0638 BAS0644 BAS0840 BAS0841 BAS0842 BAS1197 BAS1375 BAS1611 BAS1749 BAS1838 BAS1839 BAS1840 BAS2205 BAS2207 BAS2208 BAS2257 BAS3109 BAS3190 BAS3408 BAS3726 BAS4001 BAS4214 BAS4317 BAS4424 BAS4439 BAS4936 BAS4949 BAS4952 BAS5206 BAS5207 BAS5300 BAS5309 pXO1-54 pXO1-90 pXO1-122 pXO1-113

49177108 49177115 49177226 49177291 49177460 49177533 49177590 49177596 49177791 49177792 49177793 49178142 49178319 49178552 49178688 49178777 49178778 49178779 49179141 49179143 49179144 49179193 49180041 49180122 49180339 49180652 49180926 49181137 49181239 49181346 49181361 49181851 49181864 49181866 49182119 49182120 49182211 49182220 4894270 4894306 4894338 4894329

mrp protein Arginase Bacitracin ABC transporter, spermease protein, putative Iron compound ABC transporter, iron-binding protein Transcriptional regulator, Fur family Iron compound ABC transporter, iron-binding protein Immune inhibitor A metalloprotease Sphingomyelinase C CsaB protein S-layer protein Sap S-layer protein EA1 Immune inhibitor A metalloprotease Virulence factor MprF ABC transporter, substrate-binding protein, putative Enterotoxin Siderophore biosynthesis protein, putative Siderophore biosynthesis protein, putative AMP-binding protein Isochorismate synthase DhbC Isochorismatase Nonribosomal peptide synthetase DhbF Cold shock protein CspA Thiol-activated cytolysin Enhancin family protein Aconitate hydratase 1 Fibronectin/fibrinogen-binding protein, putative Transcriptional regulator, Fur family GrpE protein Substrate-binding family protein, Ferrichrome ABC transporter Iron compound ABC transporter, iron-binding protein Fe compound ABC transporter, ATP-binding protein Tyrosyl-tRNA synthetase Iron compound ABC transporter, ATP-binding protein Iron compound ABC transporter, permease protein Collagen adhesion protein, central domain Collagen adhesion protein, C-terminal Superoxide dismutase, Mn Guanosine monophosphate reductase S-layer protein S-layer protein Calmodulin-sensitive adenylate cyclase Spore germination protein XA

C C M C C C E E C CW CW E M C C C C C C C C C C M C C C C C C M C M M CW CW E C CW CW E M

MW

pI

Ames equiv. #

39.7 5.3 BA0147 32.3 4.7 BA0154 28.4 9.6 BA0284 34.5 8.3 BA0351 16.3 6.2 BA0537 36.3 9.8 BA0615 88 5.7 BA0672 42.3 6.9 BA0678 44.1 9.3 BA0884 86.6 7.6 BA0885 91.4 5.8 BA0887 86.8 5.1 BA1295 99.4 10.3 BA1486 37.4 9.7 BA1735 43.6 5.1 BA1887 69.9 5.1 BA1981 72.6 6.6 BA1982 49.4 5.8 BA1983 47 6.1 BA2369 33.4 4.6 BA2371 267.4 4.9 BA2372 7.3 4.6 BA2422 56.6 8 BA3355 85.1 6.3 BA3443 99 4.7 BA3677 64.8 9.7 BA4013 17.6 5.6 BA4313 23.4 4.4 BA4540 35.5 9.7 BA4652 36.1 8.9 BA4766 29.2 5.7 BA4784 47.3 5.3 BA5314 28.2 5.5 BA5327 37.4 11 BA5329 122.6 5.8 BA_0461 96 4.8 BA_0462 27.3 6.3 BA5696 36.2 6.6 BA5705 45 6.7 BXA0079 76.2 10 BXA0124 92.5 7.2 BXA0142 55.1 7.7 BXA0157 a C=cytoplasmic/unknown; CW=cell wall; M=membrane; E=extracellular; BA ####=B. anthracis Ames match; BA_####=B. anthracis Ames A2012 match when good Ames match not found; BXA####=B. anthracis Ames pX01 plasmid encoded proteins from A2012.

196

A.W. Francis et al. / Biochimica et Biophysica Acta 1748 (2005) 191–200

Table 2 List of surface and secreted proteins identified in this study, based on the P-sortB algorithm Gene ID

gi #

Description

Loca

MW

pI

Ames equiv.

BAS0064 BAS0130 BAS0140 BAS0182 BAS0195 BAS0196 BAS0229 BAS0247 BAS0270 BAS0283 BAS0288 BAS0472 BAS0512 BAS0525 BAS0527 BAS0541 BAS0545 BAS0547 BAS0560 BAS0609 BAS0623 BAS0638 BAS0644 BAS0653 BAS0668 BAS0669 BAS0690 BAS0704 BAS0748 BAS0757 BAS0809 BAS0828 BAS0841 BAS0842 BAS0888 BAS0916 BAS0980 BAS1021 BAS1022 BAS1025 BAS1048 BAS1050 BAS1101 BAS1102 BAS1104 BAS1105 BAS1149 BAS1197 BAS1199 BAS1214 BAS1222 BAS1225 BAS1272 BAS1301 BAS1355 BAS1363 BAS1375 BAS1518 BAS1582 BAS1591 BAS1622 BAS1680 BAS1682

49177026 49177091 49177101 49177142 49177155 49177156 49177189 49177206 49177226 49177238 49177243 49177427 49177465 49177478 49177480 49177494 49177498 49177500 49177512 49177561 49177575 49177590 49177596 49177605 49177620 49177621 49177641 49177655 49177699 49177708 49177760 49177779 49177792 49177793 49177838 49177866 49177928 49177969 49177970 49177973 49177995 49177997 49178048 49178049 49178051 49178052 49178096 49178142 49178144 49178159 49178166 49178169 49178216 49178245 49178299 49178307 49178319 49178460 49178524 49178533 49178563 49178621 49178623

Cell division protein FtsH Preprotein translocase, SecY subunit ABC transporter, ATP-binding protein Conserved domain protein Oligopeptide ABC transporter, oligopeptide-binding protein, putative Oligopeptide ABC transporter, oligopeptide-binding protein, putative Major facilitator family transporter O-sialoglycoprotein endopeptidase Bacitracin ABC transporter, spermease protein, putative Phosphoribosylformylglycinamidine cyclo-ligase SPFH domain/band 7 family protein PTS system, N-acetylglucosamine-specific IIBC component, putative Penicillin-binding domain protein SPFH domain/band 7 family protein Sensor histidine kinase Sensor histidine kinase Sensory box histidine kinase Citrate cation symporter family Glycerophosphoryl diester phosphodiesterase family protein Amino acid ABC transporter, permease protein Oligopeptide ABC transporter, oligopeptide-binding protein Immune inhibitor A metalloprotease Sphingomyelinase C Conserved hypothetical protein Quinol oxidase, subunit I Quinol oxidase, subunit II Peptidase, M23/M37 family Potassium-transporting ATPase, B subunit Na/Pi-cotransporter family protein Conserved hypothetical protein ABC transporter, ATP-binding/permease protein LPXTG-motif cell wall anchor domain protein S-layer protein Sap S-layer protein EA1 Drug resistance transporter, EmrB/QacA family S-layer protein, putative ABC transporter, permease protein EscB S-layer protein, putative Wall-associated protein, putative Wall-associated domain protein S-layer protein, putative S-layer protein, putative Oligopeptide ABC transporter, oligopeptide-binding protein Oligopeptide ABC transporter, permease protein Oligopeptide ABC transporter, ATP-binding protein Oligopeptide ABC transporter, ATP-binding protein Membrane protein, putative Immune inhibitor A metalloprotease Spermidine/putrescine ABC transporter, ATP-binding protein Sensor histidine kinase Formate/nitrite transporter family protein Small, acid-soluble spore protein, alpha/beta family ABC transporter, ATP-binding protein Potassium uptake protein, TrkH family Membrane protein, putative Penicillin-binding protein Virulence factor MprF Sodium/solute symporter family protein Flagellin Flagellar biosynthetic protein FliR Conserved hypothetical protein Membrane protein, putative N-acetylmuramoyl-l-alanine amidase, family 3

M M M M CW CW M E M M M M M E M M M M M M CW E E M M M E M M E M CW CW CW M CW M CW CW CW CW CW E M M M M E M M M E M M M E M M E M M M CW

70.1 47.5 32.6 64.4 66.7 61.6 49 36.5 28.4 37.2 31.1 53.1 30 57.9 59.9 47.4 62.1 47.9 71.9 23.6 66.5 88 42.3 18.4 73 38.6 42.5 74.7 60.3 43.5 65.5 105.9 86.6 91.4 52.3 64.8 47.2 38.5 250.3 29.4 24.8 23.6 61.6 35.1 38.1 35.7 33.7 86.8 37.8 53.1 31.3 7 26.5 49.4 42.2 76.1 99.4 54.5 32.2 28 13.1 62.2 45.3

5.7 10.9 6.3 9.4 6.5 6.7 7.6 4.9 9.6 4.9 5.3 8.1 10.4 5.2 5 8.6 8.6 9.9 9.6 9.2 7.8 5.7 6.9 9.9 7.7 8.8 10.3 5.7 5 10 5.4 4.7 7.6 5.8 9.9 9.3 10.6 9.9 6.3 10.5 7.9 4.6 7 10.5 5.7 9 9.6 5.1 6.5 5.8 7.2 9.4 5.1 10.5 9 9.5 10.3 10.3 9.6 5.8 10.2 10.2 8

BA0064 BA0130 BA0140 BA0180 BA0194 BA0195 BA0244 BA0261 BA0284 BA0296 BA0301 BA0501 BA0543 BA0557 BA0559 BA0572 BA0576 BA0578 BA0591 BA0642 BA0656 BA0672 BA0678 BA0687 BA0702 BA0703 BA0724 BA0740 BA0785 BA0796 BA0852 BA0871 BA0885 BA0887 BA0944 BA0981 BA1049 BA1093 BA1094 BA1098 BA1127 BA1130 BA1191 BA1192 BA1194 BA1195 BA1243 BA1295 BA1297 BA1313 BA1321 BA1324 BA1374 BA1409 BA1465 BA1474 BA1486 BA1635 BA1706 BA1714 BA1748 BA1815 BA1817

A.W. Francis et al. / Biochimica et Biophysica Acta 1748 (2005) 191–200

197

Table 2 (continued) Gene ID

gi #

Description

Loca

MW

pI

Ames equiv.

BAS1704 BAS1755 BAS1765 BAS1774 BAS1806 BAS1819 BAS1846 BAS1850 BAS1965 BAS1982 BAS1990 BAS2010 BAS2036 BAS2057 BAS2089 BAS2092 BAS2107 BAS2114 BAS2160 BAS2226 BAS2306 BAS2327 BAS2328 BAS2400 BAS2427 BAS2432 BAS2478 BAS2495 BAS2590 BAS2597 BAS2657 BAS2668 BAS2738 BAS2780 BAS2792 BAS2819 BAS2834 BAS2854 BAS2931 BAS2933 BAS2939 BAS3021 BAS3027 BAS3034 BAS3066 BAS3089 BAS3093 BAS3157 BAS3190 BAS3218 BAS3263 BAS3300 BAS3407 BAS3441 BAS3463 BAS3466 BAS3516 BAS3563 BAS3589 BAS3594 BAS3608 BAS3616 BAS3643

49178645 49178694 49178704 49178713 49178745 49178758 49178785 49178789 49178903 49178920 49178928 49178948 49178974 49178995 49179027 49179030 49179045 49179052 49179096 49179162 49179242 49179263 49179264 49179335 49179362 49179367 49179413 49179430 49179524 49179531 49179591 49179601 49179671 49179713 49179725 49179752 49179767 49179787 49179864 49179866 49179872 49179953 49179959 49179966 49179998 49180021 49180025 49180089 49180122 49180150 49180195 49180232 49180338 49180372 49180393 49180396 49180445 49180491 49180517 49180522 49180536 49180544 49180569

Conserved hypothetical protein Nucleoside transporter NupC Peptidase, M23/M37 family Membrane protein, putative Transport ATP-binding protein CydD Oxidoreductase, short-chain dehydrogenase/reductase family Transporter protein Phospholipase, putative Quinone oxidoreductase ABC transporter, ATP-binding protein Nitrate transporter ABC transporter, ATP-binding protein Conserved hypothetical protein Sensor histidine kinase TatCD protein Conserved hypothetical protein Mechanosensitive ion channel family protein Hypothetical protein S-layer protein, putative Hydrolase, alpha/beta fold family ABC transporter, permease protein Penicillin-binding protein, C-terminal Beta-lactamase Penicillin-binding protein, putative ABC transporter, ATP-binding protein Lipase, putative Alkaline d-peptidase Oxalate:formate antiporter, putative Oxidoreductase, short-chain dehydrogenase/reductase family Glycine betaine/l-proline ABC transporter, ATP-binding protein Oligopeptide ABC transporter, oligopeptide-binding protein, putative Sensor histidine kinase SrrB, putative ABC transporter, ATP-binding protein Nucleoside transporter, NupC family Conserved hypothetical protein Alkaline d-peptidase ABC transporter, ATP-binding protein Conserved hypothetical protein Two-component sensor histidine kinase, N-terminus Sodium/alanine symporter family protein, C-terminus 5V-Nucleotidase, putative LPXTG-motif cell wall anchor domain protein Sensor histidine kinase Major facilitator family transporter Serine/threonine transporter family protein S-layer protein, putative S-layer protein, putative ABC transporter, permease protein Enhancin family protein Oxidoreductase, short chain dehydrogenase/reductase family Drug resistance transporter, Bcr/CflA family Glycerophosphoryl diester phosphodiesterase, putative ABC transporter, ATP-binding/permease protein Urocanate hydratase N-acetylmuramoyl-l-alanine amidase, family 2 ABC transporter, ATP-binding/permease protein Hypothetical protein PTS system, fructose-specific IIABC component Membrane protein, putative Sensory box/GGDEF family protein Sulfatase Bacteriocin ABC transporter, ATP-binding protein, putative Lipoprotein, Bmp family

M M E M M E M M M M M M E M M M M E CW M M M E M M E E M E M CW M M M M E M M M M CW CW M M M CW CW M M E M E M M CW M M M M M M M M

26.6 42.5 61.3 61.7 64.6 25.5 25.7 37 35.4 32.5 42.6 72.2 10.8 51.8 28.7 38.5 34.3 23.5 42.8 39.1 88.4 65.3 34 54.1 30.7 44.7 42.2 43.3 33 45.1 62.1 52.7 61 43.8 11.2 42.1 33.3 37.6 25.8 40.9 57.9 42.1 52.5 51.4 48.3 37.6 40.3 77.2 85.1 26.1 43.1 36 63.8 60.8 66 67.1 31.4 65.8 27.4 104.1 74.2 25.9 38.4

8.4 8.4 10.5 9.3 7.4 5 10 9.1 6.6 6.5 10.2 4.9 5 9.2 8.2 9.8 5.2 10.5 7.9 5.9 10.2 6 10.2 10.1 7.5 7.3 8.6 10.6 10.2 6 9.3 6.9 5 8.8 10.4 10 6.4 9.9 10 10.1 6.5 9.6 6.2 10.3 10.1 9.8 6.1 9.6 6.3 6.3 10.2 9.8 7.5 5.1 5.3 8.8 6.6 7.3 10 7.1 5.6 9.1 9.2

BA1838 BA1893 BA1903 BA1912 BA1946 BA1958 BA_2490 BA1992 BA2113 BA2130 BA2138 BA2164 BA2191 BA2213 BA2243 BA2247 BA2263 BA2661 BA2315 BA2392 BA2481 BA2637 BA2507 BA2575 BA2603 BA2607 BA2661 BA2680 BA2778 BA2786 BA2848 BA2861 BA2948 BA2991 BA3004 BA3033 BA3048 BA3068 BA_3657 BA_3660 BA3162 BA3254 BA3260 BA3267 BA3306 BA3332 BA3338 BA3405 BA3443 BA3471 BA3518 BA3560 BA3676 BA3711 BA3737 BA3740 BA3798 BA3846 BA3873 BA3879 BA3895 BA3903 BA3927

(continued on next page)

198

A.W. Francis et al. / Biochimica et Biophysica Acta 1748 (2005) 191–200

Table 2 (continued) Gene ID

gi #

Description

Loca

MW

pI

Ames equiv.

BAS3645 BAS3729 BAS3764 BAS3793 BAS3938 BAS3960 BAS4009 BAS4035 BAS4175 BAS4177 BAS4184 BAS4203 BAS4252 BAS4257 BAS4306 BAS4333 BAS4412 BAS4413 BAS4428 BAS4431 BAS4439 BAS4486 BAS4497 BAS4560 BAS4603 BAS4638 BAS4643 BAS4671 BAS4690 BAS4693 BAS4723 BAS4730 BAS4752 BAS4798 BAS4851 BAS4882 BAS4925 BAS4949 BAS4952 BAS5033 BAS5034 BAS5043 BAS5085 BAS5090 BAS5109 BAS5206 BAS5207 BAS5240 BAS5275 BAS5281 BAS5300 BAS5330 pXO1-54 pXO1-90 pXO1-122 BXA0147a pXO1-113

49180571 49180655 49180690 49180719 49180863 49180885 49180934 49180960 49181099 49181101 49181108 49181126 49181175 49181180 49181228 49181255 49181334 49181335 49181350 49181353 49181361 49181408 49181419 49181481 49181523 49181556 49181561 49181589 49181608 49181611 49181641 49181648 49181670 49181715 49181768 49181798 49181840 49181864 49181866 49181946 49181947 49181956 49181998 49182003 49182022 49182119 49182120 49182152 49182187 49182193 49182211 49182241 4894270 4894306 4894338 20520210 4894329

Stage III sporulation protein E Conserved hypothetical protein Phospho-N-acetylmuramoyl-pentapeptide-transferase Prophage LambdaBa02, tape measure protein, putative Membrane protein, putative PTS system, glucose-specific IIABC component 5V-Nucleotidase family protein Hypothetical protein Penicillin-binding protein Superoxide dismutase, Mn Membrane protein, putative HDIG domain protein Minor extracellular protease VpR Conserved hypothetical protein Protein-export membrane protein SecDF ABC transporter, permease protein Succinate dehydrogenase, iron–sulfur protein Succinate dehydrogenase, flavoprotein subunit Conserved hypothetical protein Bacitracin ABC transporter, ATP-binding protein Iron compound ABC transporter, ATP-binding protein Malate dehydrogenase Malate dehydrogenase, putative Acetyl-CoA synthetase Polysaccharide biosynthesis family protein Permease, putative ABC transporter, ATP-binding protein Conserved hypothetical protein Cytochrome d ubiquinol oxidase, subunit I S-layer protein, putative ABC transporter, ATP-binding protein Permease, putative 1,4-Dihydroxy-2-naphthoate octaprenyltransferase, putative Cell surface protein, Gram positive anchor ABC transporter, ATP-binding protein Methyl-accepting chemotaxis protein Sodium/alanine symporter family protein Iron compound ABC transporter, ATP-binding protein Iron compound ABC transporter, permease protein Cell division ABC transporter, permease protein FtsX Cell division ABC transporter, ATP-binding protein FtsE Endopeptidase lytE, putative Nucleoside transporter, NupC family Conserved domain protein Phosphoglycerate transporter family protein Collagen adhesion protein, central domain Collagen adhesion protein, C-terminal d-Alanyl-d-alanine carboxypeptidase, putative Membrane protein, putative ABC transporter, ATP-binding protein Superoxide dismutase, Mn Mechanosensitive ion channel family protein S-layer protein S-layer protein Calmodulin-sensitive adenylate cyclase Hypothetical protein Spore germination protein XA

M M M M M M CW M M E M M E M M M M M M M M E M M M M M M M CW M M M CW M M M M M M M E M E M CW CW M M M E M CW CW E E M

88.4 16.6 35.5 104.5 28.9 76.3 58 21.7 80 22.6 32.2 80.1 100.2 14.2 82.6 73.1 29.2 65.9 25.4 34.6 29.2 33.5 46.6 64.3 60.4 43.8 27.4 64 51 29.9 28.9 75 36.6 225.2 29 73.8 51.3 28.2 37.4 35.2 25.2 47.7 42.5 33.4 48.4 122.6 96 49.2 22.6 58.8 27.3 32.1 45 76.2 92.5 6.3 55.1

6.8 10.3 10 5.4 5.3 7.3 5.6 11.1 8.5 5.4 7.2 6.9 6.1 10.1 10.3 10.2 8.5 5.9 10.5 7.2 5.7 5 5.1 5.3 10.7 9.9 5.8 5.2 10.2 10 5.8 10.2 8.9 5.3 4.5 4.9 9.8 5.5 11 10.2 5 7.6 8.4 8.7 9.8 5.8 4.8 8 8.5 5.6 6.3 5.9 6.7 10 7.2 11.1 7.7

BA3930 BA4017 BA4052 BA4082 BA4246 BA4269 BA4322 BA4350 BA4497 BA4499 BA4506 BA4528 BA4584 BA4589 BA4641 BA4668 BA4753 BA4754 BA4770 BA4774 BA4784 BA4837 BA4848 BA4915 BA4959 BA4992 BA4997 BA5031 BA5050 BA5054 BA5085 BA5090 BA5113 BA_0034 BA5217 BA5256 BA5301 BA5327 BA5329 BA5415 BA5416 BA5427 BA5475 BA5481 BA5500 BA_0461 BA_0462 BA5639 BA5672 BA5678 BA5696 BA5726 BXA0079 BXA0124 BXA0142 BXA0147 BXA0157

a

CW=cell wall; M=membrane; E=extracellular; BA ####=B. anthracis Ames match; BA_####=B. anthracis Ames A2012 match when good Ames match not found; BXA####=B. anthracis Ames pX01 plasmid encoded proteins from A2012.

solubility/location. There may be a slight bias in favour of very basic proteins with IEF and offline two-dimensional LC/MS, however this represented a small percentage of the

total. The predicted molecular weight and isoelectric point of each identified protein has been plotted as a hypothetical 2D gel to compare with the predicted 2D gel as a qualitative

A.W. Francis et al. / Biochimica et Biophysica Acta 1748 (2005) 191–200

test for bias in our methodology (Fig. 2, bottom panel). Two lobular domains exist in the predicted proteome with centroids located at pI 5 and 10. Our results (Fig. 2 top panel) display a similar overall shape. No obvious bias exists between the two plots, indicating comprehensive coverage from low to high molecular weights and pIs. A total of 17 functional groups was represented in our data (Fig. 3). dUnknownT functions are used for proteins that contain an identifiable functional domain but have not been assigned a cellular role. The category labeled dotherT represents transposon- and prophage-related proteins. A subset of the identified proteins expressed in the vegetative state that may be potentially related to pathogenesis or virulence (based on homology to proteins from other pathogens) is shown in Table 1. Although many proteins directly (or suspected to be) linked to virulence were found, two of the tripartite toxin components protective antigen (PA) and lethal factor (LF) were not detected. This could possibly be due to our growth conditions (low temp, ambient air), which are not optimal for toxin production and our sample preparation approach which would miss many secreted proteins. However we did detect a calmodulin sensitive adenylate cyclase, which is presumably edema factor (EF). Reasons for this are not clear and it suggests that there may be more virulence-related protein candidates that this study may have missed. Representation of the pXO1 plasmid, which is required for virulence, was lower than that of the chromosome (9.3% versus 19.5%). There are two reasons that could explain lower expression of the pXO1 plasmid genes: first of all, expression of the pXO1 plasmid is not required for growth, as made evident by the B. anthracis Pasteur strain (pXO1 . ) and, secondly, the plasmid contains a large number of secreted proteins (17.2% of the predicted plasmid proteome versus 2.6% of the total proteome), which would be lost in our protein isolation as mentioned earlier. 3.4. Protein localization Membrane-associated proteins (transporters, receptors, etc.), cell wall-associated proteins (adhesins, S-layer proteins, etc.), and secreted (toxins, superantigens, etc.) proteins are an important class of proteins that may be good candidates to assist vaccine [17] or antibiotic development. In this regard, a recent study by Chitlaru et al. [18] mapped immunogenic surface proteins from B. anthracis using a proteomic approach. Among the total of 1048 proteins identified in our study, 865 (82.5%) are cytosolic/ unknown, 153 (14.6%) are surface proteins (membrane- or cell wall-associated), and 30 (2.9%) are secreted proteins (compared to 4036 (73.5%) cytosolic/unknown, 1312 (23.9%) surface, and 143 (2.6%) secreted in the predicted proteome). Recent studies have shown a synergistic immunological effect upon the incorporation of an additional antigen for increased antibody generation [19,20]. To facilitate such approaches, we have compiled a list of

199

surface and secreted proteins with their locus, MW, and pI information (Table 2).

4. Discussion We have used three distinct, pre-analytical separation techniques prior to mass spectrometric analysis of the B. anthracis Sterne vegetative proteome and have identified a total of 1048 non-redundant proteins. Each separation method produced a number of unique protein identifications that were not found using the other two methods. The mass spectral data was used to search against predicted proteins from a newly sequenced B. anthracis Sterne genome which differs slightly from the recently published Ames genome. The proteins were physically and functionally classified to provide insight into the vegetative B. anthracis proteome. This proteomic analysis will facilitate data analysis from environmental samples and aid the development of improved detection techniques for B. anthracis. While the approach used in this study did not address expression levels, a comparison with analyses on related bacilli, such as B. subtilis [21], and the characterization of expressed proteins will lead to an improved understanding of the cell biology of B. anthracis and other bacilli, which may also be important in future studies aimed at the development of novel antibacterial drugs or human vaccines.

Acknowledgments We are grateful to Drs. J. Olivares and S. Gu for critical reading and discussion of the manuscript, to Dr. P. Jackson for providing the B. anthracis Sterne strain used in this work and to J. Lack for culture preparation and maintenance. This work has been conducted under support from Laboratory Directed Research and Development ER grant. Los Alamos National Laboratory is operated by the University of California for the U. S. Department of Energy under contract W-7405-ENG-36.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbapap. 2005.01.007.

References [1] T.C. Dixon, M. Meselson, J. Guillemin, P.C. Hanna, Anthrax, N. Engl. J. Med. 341 (1999) 815 – 826. [2] P. Hanna, How anthrax kills, Science 280 (1998) 1673 – 1674. [3] P. Hanna, J.A. Ireland, Understanding Bacillus anthracis pathogenesis, Trends Microbiol. 7 (1999) 180 – 182.

200

A.W. Francis et al. / Biochimica et Biophysica Acta 1748 (2005) 191–200

[4] P.C.B. Turnbull, Definitive identification of Bacillus anthracis—a review, J. Appl. Microbiol. 87 (1999) 237 – 240. [5] T. Krishnamurthy, P.L. Ross, U. Rajamani, Detection of pathogenic and non-pathogenic bacteria by matrix-assisted desorption/ionization time-of-flight mass spectrometry, Rapid Commun. Mass Spectrom. 10 (1996) 883 – 888. [6] B. Warscheid, C. Fenselau, A targeted proteomics approach to the rapid identification of bacterial cell mixtures by matrix-assisted laser desorption/ionization mass spectrometry, Proteomics 4 (2004) 2877 – 2892. [7] B. Warscheid, C. Fenselau, Characterization of Bacillus spore species and their mixtures using postsource decay with a curved-field reflectron, Anal. Chem. 75 (2003) 5618 – 5627. [8] Z. Wang, K. Dunlop, S.R. Long, L. Li, Mass spectrometric methods for generation of protein mass database used for bacterial identification, Anal. Chem. 74 (2002) 3174 – 3182. [9] H. Liu, N.H. Bergman, B. Thomason, S. Shallom, A. Hazen, J. Crossno, D.A. Rasko, J. Ravel, T.D. Read, S.N. Peterson, J. Yates, P.C. Hanna, Formation and composition of the Bacillus anthracis endospore, J. Bacteriol. 186 (2004) 164 – 178. [10] C. Redmond, L.W. Bailie, S. Hibbs, A.J. Moir, A. Moir, Identification of proteins in the exosporium of Bacillus anthracis, Microbiology 150 (2004) 355 – 363. [11] M.C. Erickson, J.L. Kornacki, Bacillus anthracis: current knowledge in relation to contamination of food, J. Food Prot. 66 (2003) 691 – 699. [12] M.L. Perdue, J. Karns, J. Higgins, J.A. Van Kessel, Detection and fate of Bacillus anthracis (Sterne) vegetative cells and spores added to bulk tank milk, J. Food Prot. 66 (2003) 2349 – 2354. [13] T. Read, S. Peterson, N. Tourasse, L. Baillie, I. Paulsen, K. Nelson, H. Tettelin, D. Fouts, J. Eisen, S. Gill, E. Holtzapple, O. Okstad, E. Helgason, J. Rilstone, M. Wu, J. Kolonay, M. Beanan, R. Dodson, L. Brinkac, M. Gwinn, R. DeBoy, R. Madpu, S. Daugherty, A. Durkin, D. Haft, W. Nelson, J. Peterson, M. Pop, H. Khouri, D. Radune, J. Benton, Y. Mahamoud, L. Jiang, I. Hance, J. Weidman, K. Berry, R. Plaut, A. Wolf, K. Watkins, W. Nierman, A. Hazen, R. Cline, C. Redmond, J. Thwaite, O. White, S. Salzberg, B. Thomason, A. Friedlander, T. Koehler, P. Hanna, A. Kolstb, C. Fraser, The genome

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

sequence of Bacillus anthracis Ames and comparison to closely related bacteria, Nature 423 (2003) 81 – 86. S. Iyer, J. Olivares, Trypsin digestion of proteins on intact immobilized pH gradient strips for surface matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis, Rapid Commun. Mass Spectrom. 17 (2003) 2323 – 2326. A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels, Anal. Chem. 68 (1996) 850 – 858. J. Gardy, C. Spencer, K. Wang, M. Ester, G. Tusna´dy, I. Simon, S. Hua, K. deFays, C. Lambert, K. Nakai, F. Brinkman, PSORT-B: improving protein subcellular localization prediction for Gramnegative bacteria, Nucleic Acids Res. 31 (2003) 3613 – 3617. N. Ariel, A. Zvi, K. Makarova, T. Chitlaru, E. Elhanany, B. Velan, S. Cohen, A. Friedlander, A. Shafferman, Genome-based bioinformatic selection of chromosomal Bacillus anthracis putative vaccine candidates coupled with proteomic identification of surface-associated antigens, Infect. Immun. 71 (2003) 4563 – 4579. T. Chitlaru, N. Ariel, A. Zvi, M. Lion, B. Velan, A. Shafferman, E. Elhanany, Identification of chromosomally encoded membranal polypeptides of Bacillus anthracis by a proteomic analysis: prevalence of proteins containing S-layer homology domains, Proteomics 4 (2004) 677 – 691. G. Rhie, M. Roehrl, M. Mourez, R. Collier, J. Mekalanos, J. Wang, A dually active anthrax vaccine that confers protection against both bacilli and toxins, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 10925 – 10930. R. Schneerson, J. Kubler-Kielb, T. Liu, Z. Dai, S. Leppla, A. Yergey, P. Backlund, J. Shiloach, F. Majadly, J. Robbins, Poly(gamma-dglutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of Bacillus anthracis: a potential addition to the anthrax vaccine, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 8945 – 8950. C. Eymann, A. Dreisbach, D. Albrecht, J. Bernhardt, D. Becher, S. Gentner, T. Tam, K. Bqttner, G. Buurman, C. Scharf, S. Venz, U. Vflker, M. Hecker, A comprehensive proteome map of growing Bacillus subtilis cells, Proteomics 4 (2004) 2849 – 2876.